Molecular cross talk between misfolded proteins in animal models of Alzheimer's and prion diseases

Abstract

The central event in protein misfolding disorders (PMDs) is the accumulation of a misfolded form of a naturally expressed protein. Despite the diversity of clinical symptoms associated with different PMDs, many similarities in their mechanism suggest that distinct pathologies may cross talk at the molecular level. The main goal of this study was to analyze the interaction of the protein misfolding processes implicated in Alzheimer's and prion diseases. For this purpose, we inoculated prions in an Alzheimer's transgenic mouse model that develop typical amyloid plaques and followed the progression of pathological changes over time. Our findings show a dramatic acceleration and exacerbation of both pathologies. The onset of prion disease symptoms in transgenic mice appeared significantly faster with a concomitant increase on the level of misfolded prion protein in the brain. A striking increase in amyloid plaque deposition was observed in prion-infected mice compared with their noninoculated counterparts. Histological and biochemical studies showed the association of the two misfolded proteins in the brain and in vitro experiments showed that protein misfolding can be enhanced by a cross-seeding mechanism. These results suggest a profound interaction between Alzheimer's and prion pathologies, indicating that one protein misfolding process may be an important risk factor for the development of a second one. Our findings may have important implications to understand the origin and progression of PMDs.

Figure 1.

Alzheimer's pathology accelerates prion disease in mice models. A, To assess the effect of AD neuropathology in the onset of prion disease, we inoculated intraperitoneally Tg2576 mice with RML prions at 365 (orange line) and 45 (green line) days of age. As control, age-matched WT mice (nontransgenic littermates) were inoculated with the same stock and quantity of the infectious agent (black line). WT animals infected at 45 or 365 d of age developed prion disease at very similar times, and thus the data for these two groups are presented together in the graph. Clinical signs were assessed as described in Materials and Methods. When animals were definitively diagnosed with prion disease, they were killed to avoid additional pain. The data show that both groups of Tg2576-inoculated mice develop prion disease in a shorter time compared with age-matched WT controls. In addition, we performed a second infectivity passage in WT mice by inoculating infectious material from the brain of a sick Tg2576 animal injected at 365 d of age (blue line). B, Average of incubation periods of the different groups shown in A, including the statistical comparison between each experimental group with the WT control mice. The statistical analysis was done using the Student t test.

Figure 2.

A–D, Brain histopathological studies. Representative animals from different groups were studied histopathologically for spongiform brain degeneration after hematoxylin–eosin staining (A), reactive astrogliosis by GFAP staining (B), Aβ deposition by immunohistochemistry using the 4G8 anti-Aβ antibody (C), and staining with the amyloid-specific dye thioflavin S (D). It is important to emphasize that the prion deposits in mice affected by RML prions are not thioflavin S positive but are rather diffuse prefibrillar aggregates. The images in A and B correspond to the medulla, C to the hippocampus or cortex as indicated, and D to the cortex. (See also supplemental Figs. 2, 3, available at www.jneurosci.org as supplemental material.)

Figure 3.

PrP^Sc levels in Tg2576 or WT mice inoculated with prions. A, Brain homogenates from clinically sick Tg2576 mice inoculated with prions at 45 or 365 d of age or, as a control, Tg2576 mice noninoculated with prions (right panel) were PK digested and Western blotted to analyze PrP^Sc burden. The result shown corresponds to one animal representative of all mice in the group. The animal of the 45 d group shown in the figure was killed at 190 dpi, and the animal of the 365 d at 159 dpi. B, For comparison, we measured the PrP^Sc levels in the brain of WT mice challenged with RML prions and killed at 140, 169, 193, and 225 dpi. Only the animal at 225 dpi was exhibiting signs of prion disease. Numbers at the top of each gel represent brain dilution. Brain dilutions were performed from a 10% brain homogenates, and various dilutions are shown to facilitate the comparisons. C, Densitometric analysis of PrP^Sc quantity in different animals. Western blot data as those shown in A and B were evaluated densitometrically to estimate the extent of the PrP^Sc signal. The data represent the average and SE of five different animals in each group. As seen, Tg2576 mice inoculated with prions at 45 and 365 d of age accumulate a similar quantity of PrP^Sc as WT mice but in a much shorter time. **p < 0.01. (See also supplemental Table 1, available at www.jneurosci.org as supplemental material.)

Figure 4.

Interaction between Aβ and PrP in the brain. A, Sections from the cortex of animals in different groups were stained with fluorescent antibodies against Aβ (4G8; green) and PrP (6H4; red), and colocalization was evaluated by confocal microscopy. The white arrows point to the typical Aβ amyloid plaques seen in transgenic mice and AD patients; the yellow arrows point to the typical diffuse accumulations of PrP^Sc aggregates in the brain of TSE-affected individuals. B, Coimmunoprecipitation experiments. Aliquots from the brain of animals from different groups were immunoprecipitated by 4G8 antibody, which specifically recognize Aβ. The immunoprecipitated material was separated by electrophoresis and analyzed by Western blot using the 6H4 anti-prion antibody and, as control, the AKT antibody.

Figure 5.

Cross-seeding of PrP and Aβ misfolding and aggregation in vitro. A, The effect of purified PrP^Sc on Aβ aggregation was measured over time by sedimentation followed by sensitive ELISA. Seed-free soluble Aβ1-42 (0.01 mg/ml) was incubated with different concentrations of purified PrP^Sc seeds or PBS (control). The concentration of PrP^Sc is expressed as a percentage of oligomers per Aβ monomer and was calculated assuming that a PrP^Sc oligomer has an average molecular weight of 7700 kDa. The latter was based on data coming from flow field fractionation of PrP^Sc and corresponds to the fraction with the highest concentration of PrP^Sc (Silveira et al., 2005). Samples were incubated at 25°C with shaking for the indicated times. Thereafter, soluble and aggregated Aβ were separated by centrifugation at 14,000 rpm for 10 min, and the quantity of peptide in the supernatant was measured by ELISA. The experiment was done by triplicate, and results represent the average ± SE. Analysis by two-way ANOVA (using condition and time as the variables) show that the kinetics of Aβ aggregation in the presence of PrP^Sc is highly significantly different from the control (p < 0.0001). B, The effect of Aβ aggregates on PrP misfolding was studied by incubating 10 μg of recombinant mouse PrP in the presence of increasing concentrations of preformed Aβ fibrils. Fibrils were prepared as indicated in Materials and Methods, and aliquots corresponding to 0.14% (1.2 μg of total Aβ), 0.28% (2.4 μg of total Aβ), 0.56% (4.8 μg of total Aβ), and 1.1% (9.6 μg of total Aβ) were added to monomeric recPrP (lines 1, 2, 3, and 4, respectively). The concentration of Aβ fibrils is expressed as a molar percentage per recPrP monomer and was calculated assuming that the average molecular weight of Aβ fibrils is 2000 kDa, as estimated by a combination of size-exclusion chromatography, atomic force microscopy, and electron microscopy (Goldsbury et al., 2000). The mixture was incubated for 30 h at 37°C in an Eppendorf Thermomixer with cycles of 1 min agitation at 1500 rpm and 1 min incubation. To assess PrP misfolding, samples were incubated at 37°C with 7 μg/ml PK and PrP^res signal analyzed by Western blot. Line 5 is the control with the same quantity of recPrP incubated in the absence of Aβ fibrils. Line 6 corresponds to recPrP nontreated with PK to display the migration of the full-length protein. The numbers at the right side correspond to the molecular weight standards.